The long term goal of this research is to understand the basis for genomic heterogeneity in the efficiency of nucleotide excision repair and the factors that regulate DNA repair in mammalian cells. Current models of DNA repair in mammalian cells presume that repair of DNA damage is faster on the transcribed strand than the nontranscribed strand of actively expressed genes, and that preferential repair of damage on the transcribed strand is directly coupled to transcription. However, recent findings have raised serious questions concerning the universality of such models. Rapid repair of CPD on both DNA strands of this region is also seen in an APRT promoter-deletion mutant in which there is no transcription of APRT gene sequences. However, in a second APRT promoter-deletion mutant with a much larger 5'-extending deletion, CPD on both strands are very inefficiently repaired. Our findings appear to challenge current dogma that preferential repair of DNA damage in actively expressed genes in simply a consequence of transcription-coupled repair of transcription- blocking lesions, and suggest that other mechanisms may be important in controlling nucleotide excision repair in mammalian cells. We have recently discovered another, convergently transcribed gene just downstream of APRT gene. We propose to determine whether the rapid repair of CPD on both DNA strands observed at the APRT locus is dependent upon the strand-specific, transcription-coupled repair of multiple closely-spaced, actively- transcribed genes in this region, or is independent of transcription. We hypothesize that the striking differences in CPD repair observed between our two different APRT promoter-deletion mutants might be a consequence of either the deletion of an upstream cis-acting control element normally responsible for maintaining an "open" chromatin configuration that facilitates efficient repair of the region, or the deletion-mediated juxtaposition of chromosomal regions resulting in "position effects" that can dramatically affect the efficiency of CPD repair over a large genomic domain. Finally, we propose to generate, by targeted gene replacement, DNA repair-proficient and -deficient CHO cell lines in which the endogenous APRT gene has been "flipped" with respect to its original chromosomal/transcriptional orientation. These cell lines will allow assessment of the contributions of strand-specific transcription-coupled repair and differential fidelity of leading/lagging strand synthesis to the strand-bias of UV-induced mutation at the APRT locus.